Oxygen uptake by the lung is governed by local matching of alveolar ventilation (VA) to blood flow (perfusion, Q). VA/Q matching is mediated by pO2-dependent regulation of smooth muscle tone in blood vessels where arterial pressure reflects operation of the physiological response, hypoxic pulmonary vasoconstriction (HPV). Although pO2 can directly regulate smooth muscle, the cellular mechanisms of VA/Q matching have not been fully elucidated. The central theme of our research is the emerging role of red blood cells (RBCs) in the control of O2 uptake at the lung-blood interface. We have demonstrated that RBCs regulate HPV and thus pulmonary artery pressure, and improve oxygenation, and that these effects are mediated through the pO2- dependent formation of S-nitroso-hemoglobin (SNO-Hb) and subsequent delivery of vasodilatory nitric oxide (NO) equivalents. In addition, we have found, surprisingly, that the delivery of NO-related bioactivity from SNO-Hb improves ventilation, at least in part by dilating airways. Thus, the central hypothesis of this proposal is that the pO2-regulated generation and delivery of bioactive NO equivalents by RBCs plays a significant role in VA/Q matching, by regulating both ventilation and perfusion of alveolar units. To elucidate the role of RBCs in VA/Q matching and the enzymatic mechanisms that govern the formation and delivery of NO-related bioactivity in the lung, we have developed murine and rabbit models of hypoxic pulmonary hypertension, in which SNO-Hb levels are manipulated using genetic and biochemical approaches. We will use these models to test the specific hypotheses that: 1) deficiency or excess of SNO-Hb interferes with optimal gas exchange within the lung by disrupting VA/Q matching; 2) the enzymatic source of NO equivalents required for SNO-Hb formation (and thus for optimal blood oxygenation) is eNOS (and therefore the role of eNOS in VA/Q matching is carried out in significant part through the agency of RBCs); 3) SNO-Hb levels within RBCs (and thereby O2 uptake by the lung) are critically regulated by the enzyme S-nitrosoglutathione reductase; 4) the delivery of NO-based bioactivity by RBCs entails an essential role for gamma-glutamyl transpeptidase. Understanding how RBCs regulate the coordinated pulmonary vascular and airway responses that optimize gas exchange should facilitate novel diagnostic and therapeutic approaches to lung dysfunction, including acute lung injury, transfusion-related morbidity and chronic hypoxemic lung disease, and also point to potential roles of RBC- derived vasoactivity in other disorders that are characterized by tissue hypoxemia (e.g., sepsis and heart failure). PUBLIC HEALTH RELEVANCE: Red blood cells (RBCs) contain vasodilatory S-nitrosothiols (SNO) and have been ascribed a novel role in dispensing nitric oxide bioactivity. We have recently shown that patients with pulmonary hypertension have a deficiency of RBC SNO that impairs RBC vasodilation, and that repletion of SNO is associated with improvements in both RBC bioactivity and pulmonary function. Here we employ pharmacologic and genetic approaches to explore the possibility that RBCs can modulate both pulmonary vascular and airway tone, thereby optimizing gas exchange (oxygen uptake), and we offer new molecular insights that may broadly impact the diagnosis and treatment of heart, lung and blood diseases, including sepsis, heart failure, and pulmonary arterial hypertension.